Anthelmintic hygromycin b derivatives and process of using same



United States Patent 3,293,128 ANTHELMINTIC HYGROMYCIN B DERIVATIVES ANDPROCESS OF USING SAME Robert L. Mann, Indianapolis, Ind., assignor toEli Lilly and Company, Indianapolis, Ind., a corporation of indiana NoDrawing. Filed Nov. 24, 1961, Ser. No. 154,890 7 Claims. (Cl. 16765)This invention relates to new therapeutic agents useful for thetreatment of parasitic infections and to processes for treating suchinfections. More particularly, this invention relates to N -substitutedderivatives of hygromycin B and the acid addition salts thereof, theirprepa ration, and their use in the treatment of helminthiasis.

Helminthiasis is one of the most serious and widely prevalent problemsin the raising of farm animals. The economic losses brought about byparasitic infections of animals grown commercially are staggering. Theselosses occur not only as the result of extremely severe parasiticinfections which cause death of the animals, but also as the result ofinsidious infections which by their debilitating effects reduce feedefificiency and carcass quality and increase the susceptibility of theanimals to other disease conditions. The problem is likewise serious inthe case of house pets where the general health of the animal isfrequently impaired by a parasitic infection.

One of the most effective therapeutic agents currently in use for thetreatment of helminthiasis in animals is hygromycin B, an antibioticdiscovered by McGuire and Mann and described in United States patentapplication Serial No. 690,399, filed October 15, 1957, now U.S. PatentNo. 3,018,220. It has the structure represented by the formula HO OHwherein Z is a sugar moiety whose structure has not yet been completelyelucidated, and wherein the nitrogen atoms are convenientlydistinguished as N in the primary amino group and N in the secondaryamino group.

Hygromycin B has been utilized extensively as such in the treatment offarm animals, especially swine and poultry. When it is employed over aprolonged period of time, however, it occasionally produces animpairment of hearing in some animals, particularly swine, which appearto be especially susceptible. Although the problem occurs onlyinfrequently when the hygromycin B is fed at the recommended dosages forthe recommended time, it does discourage the use of the antibiotic in acontinuous feeding program. Thus, a substance which would retain thehighly desirable anthelmintic properties of hygromycin B and at the sametime have reduced toxicity would be a valuable addition in the field ofanthelmintic therapy.

It is an object of this invention to provide new anthelmintic substancesof low toxicity having a high order of efficacy. A further object is toprovide a process for the preparation of such substances. Still anotherobject of this invention is to provide methods for the treatment ofhelminthiasis in animals. Other objects will become apparent from thespecification and claims which follow.

The compounds of the present invention are N -substituted hygromycin Bderivatives represented by the formula R3 HgC-NH NH-ft -R Ho OH alkoxy,or di(lower)alkylamino; and R is hydrogen, aryl,

or heterocyclic containing 0, S, and/or N; wherein the aryl group can beadditionally substituted by amino, hydroxy, lower alkoxy,di(lower)alkylamino, halogen, or methylenedioxy; and wherein theheterocyclic group'can be additionally substituted by amino, loweralkyl, lower alkoxy, di(lower)alkylamino or halogen.

Among the examples of aryl groups defined by R are phenyl,hydroxyphenyl, methoxyphenyl, dimethylaminophenyl, chlorophenyl,br-omophenyl, fluorophenyl, salicyl, vanillyl, dichlorophenyl,dimethoxyphenyl, naphthyl, and the like.

R when it defines a heterocyclic group, can be carbazolyl, dioxanyl,furyl, imidazolyl, indolyl, isoindolyl, isoquinolyl, isoxazolyl,oxazolyl, pyrazinyl, pyrazolyl, pyridyl, pyrimidyl, pyrryl, quinazolyl,quinolyl, quinoxalyl, thiazolyl, thienyl, triazolyl, and the like; thepar-. tially and completely hydrogenated derivatives thereof; thederivatives thereof having alkoxy, alkyl, amino, dialkylamino, halogen,or hydroxy substituted at one or more positions on the ring, as forexample, the pioolyls, methylfuryls, methylthienyls, bromopyridyls, andthe like; and heterocyclic radicals having a fused benzo ring, such asbenzodioxanyl, benzofuryl, benzothienyl, and the like.

In accordance with the invention, N -substituted derivatives ofhygromycin B having a high degree of anthel-. mintic eflicacy areprepared by allowing an aldehyde to react with hygromycin B to form anintermediate of the Schilfs base type, and reducing the intermediate bya suitable catalytic or chemical means. It is not necessary to isolatethe intermediate Schifis base prior to reduction, although this may bedone if desired. Reduction of the intermediate can be effectedcatalytically at temperatures of from about 20 to about C. and hydrogenpressures of about 30 to about 1500 p.s.i. with catalysts such asplatinum oxide, 5% palladium on carbon, 5% palladium on alumina, 5%rhodium on carbon, and the like. Alternatively, the reduction can becarried out with sodium borohydride in aqueous or alcoholic solutions.Numerous alternative reduction techniques will be readily apparent tothose skilled in the art. Aldehydes used in this process can bealiphatic, aromatic, or heterocyclic and are limited only by theiravailability. Illustrative of the aldehydes which can be used areacetaldehyde, crotonaldehyde, glycolaldehyde, undecylenic aldehyde,4-acetaminobenzaldehyde, 2-chlorobenzaldehyde, 3-methoxybenzaldehyde,4-nitrobenzaldehyde, 3,4,5-trimethoxybenzaldehyde, veratraldehyde,Z-naphthaldehyde, furfural, 2-thiophenecarboxaldehyde, and the like, andin general virtually any aliphatic, aromatic, or heterocyclic aldehyde.

The N'-substituted hygromycin B derivatives and their physiologicallyacceptable salts are employed for the control of helminthiasis byadministering to a parasitized host an effective amount of the drug,usually between about 100 mg. per kg. and 1000 mg. per kg. of host bodyweight, preferably between about 250 mg. per kg. and 750 mg. per kg. Theselected derivative can be supplied in any of a variety of dosage formswhich may include the drug alone or in combination with a pharmaceuticalexcipient such as a solid or liquid diluent, bulfer, binder, coatingmaterial, preservative, emulsifier, or the like. The solid dosage formsare especially convenient to administer and may in one embodiment of theinvention consist of the selected compound incorporated in aphysiologically compatible excipient, for example an edible feedstuif,in such proportion as to provide the drug in high concentration, inwhich arm it may be added to or mixed with the animals feed 1 the properdosage amount. The dose required for the nimal can be blended with aportion or all of the feed onsumed by the animal during a day or othertime interal. The term edible feedstuff as used in this disclosure iayrefer to a normal dietary constituent of the animal, uch as a completeration, or a component or combination f components of the completeration, or a mineral or itamin supplement or the like, and in generalany dietary omponent which is physiologically utilizable by the aniial.Alternatively, the excipient can be any bland, nonritating materialwhich will be accepted by the animal, ut which itself is notphysiologically utilizable, as for exmple an ion exchange resin or thelike.

Other solid dosage forms such as tablets and/r filled apsules comprisingthe anthelmintic agent and one or 1ore of the commonly used diluentssuch as talc, lactose, tarch, magnesium stearate, methylcellulose, andthe like an be employed with equally good results.

For the treatment of helrninthiasis in farm animals, the ompounds ofthis invention are conveniently adminisared by incorporating them withthe normal feed conumed by the animals. The concentration of theanthelnintic agent in the feed can vary from about 5 g. per ton o about50 g. per ton, being preferably about 25 g. per on. The animals arepermitted to consume the medicated eed for varying periods of time,depending upon the effect .esired. Thus, animals which are heavilyparasitized can e treated for a short time with a feed containing arelaively high proportion of the drug, for example 35 to 40 g. Ier tonof feed, until the existing parasite burden is elimuated orsatisfactorily reduced. Thereafter, the animals an be fed coninuously aration having a lower drug conentration, as for example to g. per ton offeed, [1 order to maintain them in a deparasitized condition. )thervariations in drug concentrations and treatment chedules will beapparent to those skilled in the art. The 'elatively low toxicity of theN -substituted hygromycin B lerivatives makes possible their use in aprolonged feeding :rogram without serious side effects. This lack oftoxicity ias been demonstrated in the case of N -benzylhygromy- :in B byfeeding the drug in concentrations as high as 240 g. per ton for as longas 16 weeks without any adverse :ifect on growth or feed efliciency andwith no apparent mpairment of hearing.

The compounds of the present invention are stable, vhite or nearlywhite, amorphous solids of indefinite meltng point. Their infraredabsorption spectra are, in gen- :ral, not sufiicient distinguished fromthe spectrum of hygromycin B to serve to characterize the compounds.Jltraviolet absorption spectra are of value only in the case if thosecompounds derived from aldehydes having aronatic character. These, ofcourse, exhibit the absorption :haracteristics of the aromatic nucleus.The compounds lre characterized primarily by microbiological assaynethods, bioautograph, and thin-layer chromatography. [he intermediateSchiffs bases show the expected C=N absorption in both the infrared andultraviolet 'egions. This is most useful as a means for following thearogress of the reduction of the Schifis bases, since the ntensity ofabsorption diminishes as the saturation of the C=N bond proceeds.

The general procedures used in the preparation of the :ompounds of thisinvention and some of the methods of itilizing them in the treatment ofhelminthiasis are illus- :rated in the examples which follow. Theinvention, how- :ver, is not to be construed as being limited to thesemethids, either preparative or therapeutic, inasmuch as other variationswill be apparent to those skilled in the art.

Example 1 To a suspension'of 4 g. of hygromycin B in 15 ml. of absoluteethanol there were added about 1.6 molar equivilents of furfural. Themixture was shaken occasionally intil essentially all of the materialwas in solution, and was then filtered into 100 ml. of ether. Theintermediate precipitated thereby was removed by filtration, washed withether, and air dried. The dry intermediate was dissolved in 30 ml. ofmethanol and cooled in an ice bath while a solution of 800 mg. of sodiumborohydride in 15 ml. of cold methanol was added. The reaction mixturewas kept inv the ice bath until gas evolution had almost ceased and wasthen allowed to warm to room temperature. After being heated for onehour at C. on a Water bath, the solution was filtered into 300 ml. ofether. The precipitated N -furfurylhygromycin B was removed byfiltration, washed with ether, and air dried.

Example 2 The procedure of Example 1 was followed in detail except thatthe addition of the sodium borohydride solution was made directly to thesolution of the Schiffs base with out preliminary isolation of theintermediate. The product was identical to that obtained in the previousexample.

Example 3 To a solution of 4 g. of hygromycin B in 20 m1. of water therewere added 1.6 ml. of beuzaldehyde. The reaction mixture was shakenfrequently for 2 /2 hours, after which it was cooled in an ice bathwhile 800 mg. of solid sodium borohydride were added with care. Themixture was allowed to warm to room temperature and kept withoutadditional heating for another hour. The reaction mixture was filteredinto acetone and the precipitated N -benzylhygromycin B was removed byfiltration, washed with acetone and ether, and air dried.

Example 4 Except for solvent composition, the procedure of Example 3 wasfollowed in detail. The effect of varying the composition of the solventused to'dissolve the hygromycin B was determined. Methanol-water ratiosof 9:1, 4:1, 7:3, 3:2, 2:3 and 1:9 were employed without ap-- parenteffect on the course of the reaction.

Example 5 A mixture of 4 g. of hygromycin B, 15 ml. of ethanol and 2 ml.of benzaldehyde was stirred at room temperature until complete solutionoccurred. Suflicient ethanol was added to give a total volume of 150 ml.After addition of 2 g. of 5% palladium on carbon, the mixture was shakenunder a hydrogen pressure of 500 psi. for

- eight hours at 50 C. After removal of the catalyst by filtration, thefiltrate was concentrated to a small volume and poured into ether. Theprecipitated N -benzylhygrornycin B was filtered, washed with ether, andair dried. The product was identical to that obtained by the methodsdescribed in the previous examples.

Example 6 The general procedure of Example 5 was followed using platinumoxide as a catalyst under an initial hydrogen pressure of 41 p.s.i. Thereduction took place at ambient temperature during twenty hours. The N-benzylhygromycin B obtained in this way was indistinguishable from thatobtained by the other described methods.

Example 7 receiving the basal ration without drug served as a control ineach case. All parasites passed during the treatment period were counteddaily. At the end of the experiment the animals were sacrificed and theunexpclled worms were counted. The sum of the passed worms and theremaining worms constituted the total worm burden for each animal. Theresults with N -furfurylhygrornycin B are shown in Table 1, those with N-heptylhygrornycin B in Table II.

TABLE I.AN'IHELMINTIC EFFICACY IN SWINE OF N -FURFURYLHYGROMYCIN B WormsEliminated 1 Drug Concentration No. of Pigs Ascaris Oesophagostumumsuum, percent app, percent Control 5 48 2 24 gJton- 6 71 75 3D g./t0n 448 69 36 gJton. 2 98 91 48 g.lton 2 100 100 1 lzercent Worms Eliminatedworms passed/total worms X 100%.

TABLE II.ANTHELMIN'IIO EFFICACY IN SWINE OF N HEPTYLHYGROMYCIN B WormsEliminated 1 Drug Concentration N or of Pigs Ascaris Oesophagosiumumsuum, percent spp., percent 1 As in Table I.

Example 8 The procedure of Example 7 was followed in detail with N'benzylhyg-romycin B except that the treatment period was fifty-sixdays. The results are given in Table III.

TABLE III.ANTHELMINTIC EEETCACY I'N s'wINE or NLBENZYLHYGROMYCIN B WormsEliminated 1 Drug No. of I Concentration Pigs Ascaris Oesophagostfl-Trichuns suu'm, mum spp, suis, percent percent percent Control 4 32 1637 12 g./t0n 4 23 85 18 g./ton 4 56 88 94 24 g./ton 4 14 100 67 30g./ton 4 88 97 59 36 g./ton 4 95 98 64 1 As in Table I.

Example 9 The general procedure of the previous examples was followedusing combinations of hygromycin B and N benzylhygromycin B. Thetreatment period was fifty-six days. The results are presented in TableIV. All treated groups received 6 g. per ton of hygromycin B and theindicated amount of N benzylhygromycin B.

TABLE IV.ANTHELMINTIC EFFICACY IN SWINE OF MIXTURES OF HYGROMYCIN B ANDN -BENZYLHYDRO- MYCIN B Worms Eliminated I Concentration of N -B enzyl-N0. of hygromycin B Pigs Ascaris Oesophaflostum- Trtchuria suum, um app,suis, percent percent percent Control 6 41 3 27 6 g./ton 6 100 95+ 99+ 8g./t0n 6 100 99+ 95+ 10 g./t0n 6 100 100 1 As in Table I.

Example 10 Evaluation of the compounds for anthelrnintic activity wasalso carried out in mice against the mouse pinworm Syphacia obvelata, aspecies generally considered by those E-dimethylaminopival Benzaldehydep'Fluorobenzaldehyde 407 63 99 p-Dimethylaminobenzaldehyde- 657 86 94418 94 Salicylaldehyde 402 99 Vanillin- 414 75 99 Piperonal 391 100a-Methylcinnamaldehyd 232 0 99 Furiural 317 100 100 210 75 99fi-methyl-Z-pyridylaldehyde. 318 100 100 Indoie-3-carboxaldehyde 288 100100 1 Average calculated drug intake per mouse. small quantities duringa week.

This may exceed LD because taken in 2 Percent Ciearance=Number of micecompletely free of worms/Number of mice on test 100%.

3 Percent Reduction=Ave. N0. Mice per control animal less ave. No. miceper treated animal/Ave. No. mice per control animal 100%.

Acute toxicity determined orally in mice. Approx. LD for hygromycin B=75:killed in the art to parallel the response of other helminth pecies,especially Enterobius vermicularis. Mice natually infected with Syphaciaobvelata were kept for one veek on a diet into which was incorporated agiven per :entage by weight of the drug being tested. Similar groups ofnaturally infected mice were kept as untreated :ontrols. At the end ofone week, all mice were sacrificed llld the parasites remaining werecounted. Comparison 1f the worm burdens of the treated and control micearovidcd a measure of the worm reduction effected by the lrug. Theanthelmintic efficacy and oral toxicities for a representative group ofcompounds appear in Table V.

I claim:

1. The N -substituted hygromycin B derivatives having he formula [ridthe salts thereof with pharmaceutically acceptable lCldS, wherein:

Z represents the sugar moiety of hygromycin B; R is a saturated orolefinic (l -C aliphatic hydrocarbon connecting group; R is hydrogen,hydroxy, lower alkoxy or di (lower) alkylamino; and R is hydrogen;phenyl; naphthyl; phenyl having at least one substituent, thesubstituents being amino, hydroxy, lower alkoxy, di(lower) alkyamino,halogen,

or methylenedioxy; or a heterocyclic group containing at least onehetero atom of the group consisting of O, S, and N.

2. N -benzylhygromycin B.

3. N -furfurylhygromycin B.

4. N -heptylhygromycin B.

5. The process of treating helminthiasis in animals which comprisesorally administering to the host animal a therapeutic quantity of thecompound of claim 1.

6. The process of treating helminthiasis in animals which comprisesfeeding to an infected animal a medicated feed containing as an activeingredient thereof the compound of claim 1, said active ingredient beingpresent in the feed in a concentration of between about 5 g. per ton andabout 50 g. per ton.

7. The process of treating helminthiasis in animals which comprisesfeeding to a helminth infected animal a medicated feed containinghygromycin B in a concentration between about 3 g. per ton and about 8g. per ton of feed and the composition of claim 1 in a concentration ofbetween about 5 g. per ton and about 20 g. per ton.

References Cited by the Examiner Fieser and Fieser: Organic Chemistry,2nd ed. (1950), p. 638.

JULIAN S. LEVITT, Primary Examiner.

SAM ROSEN, Assistant Examiner.

1. THE N1-SUBSTITUTED HYGROMYCIN B DERIVATIVES HAVING THE FORMULA